Galf-Specific Neolectins: Towards Promising Diagnostic Tools

In the absence of naturally available galactofuranose-specific lectin, we report herein the bioengineering of GalfNeoLect, from the first cloned wild-type galactofuranosidase (Streptomyces sp. strain JHA19), which recognises and binds a single monosaccharide that is only related to nonmammalian species, usually pathogenic microorganisms. We kinetically characterised the GalfNeoLect to confirm attenuation of hydrolytic activity and used competitive inhibition assay, with close structural analogues of Galf, to show that it conserved interaction with its original substrate. We synthetised the bovine serum albumin-based neoglycoprotein (GalfNGP), carrying the multivalent Galf units, as a suitable ligand and high-avidity system for the recognition of GalfNeoLect which we successfully tested directly with the galactomannan spores of Aspergillus brasiliensis (ATCC 16404). Altogether, our results indicate that GalfNeoLect has the necessary versatility and plasticity to be used in both research and diagnostic lectin-based applications.


Introduction
Glycans, oligosaccharide moieties that originate from a small set of monosaccharides, are typically found conjugated to glycoproteins and glycolipids and are exposed to the extracellular side of membranes, where they mediate a tremendous variety of cell interactions and signalling [1][2][3].Among the putative set of monosaccharide constituents of glycans in organisms, galactofuranose (Galf ), the five-membered ring form of aldohexose galactose, is completely absent from mammalian glycan motifs, including humans.Galactofuranose is by far the most widespread in other domains of life, ranging from archaea and bacteria to protozoa, fungi, and plants [4][5][6][7][8].Often found in pathogenic organisms such as the bacterium Mycobacterium tuberculosis, the fungus Aspergillus fumigatus, and the protozoan Leishmania major as examples, it is hypothesised to be an advantageous element for their survival, reproduction, and virulence in the host [9].As such, Galf -deficient mutants in Aspergillus have significant modifications of the cell surface that result in aberrant morphological changes and growth reduction, whilst the virulence capacity of Leishmania species is reduced [10].Therefore, exploiting the presence of Galf in the glycome of pathogenic microorganisms makes it a promising target for biomedical research since it circumvents the potential risk of interference with the human glycans [11].The distinctiveness of Galf as a component of important surface glycoconjugates of human pathogens has led to increased interest in targeting it in diagnostic or imaging techniques.Recent diagnostic techniques are predominantly based on production of Galf -specific antibodies.The presence of Aspergillus exoantigens of galactomannan (GM) origin is a specific indicator of invasive pulmonary aspergillosis [12].Monoclonal antibody detection methods for early serological diagnosis of GM antigens of invasive pulmonary aspergillosis have been experimentally developed since the 1980s.In 1995, a double-direct sandwich enzyme-linked immunosorbent assay (ELISA), that uses a rat anti-GM monoclonal antibody (EB-A2) directed against the β-Galf -(1 → 3)-β-Galf epitopes of GM, was developed.Today, two GM antigen-detection kits are commercially available, the ELISA Pastorex ® Aspergillus test (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) and the latex agglutination test (LAT) Platelia ™ Aspergillus (Biorad, Marnes-la-Coquette, France) [10].The bibliographical overview of different types of Galf -specific antibodies is summarised in Table 1.research study [13,14] Polyclonal inhibitory antisera-rabbit Ab IgG β-D-galactofuranosyl units of surface glycoproteins Trypanosoma cruzi research study [15] Human serum monoclonal Ab (L-5-27) -suggested it recognises terminal α-Galp-(1,3)-β-Galf epitope Leishmania major research study [16] Rabbit serum -Galf -containing LPPG & LPGG-like molecules Trypanosoma cruzi research study [17] Rat monoclonal Ab (EB-A2) IgM (1,5)-β-Galf -containing epitope of the galactomannan molecule Aspergillus fumigatus research study; diagnostics (Platelia ®  ELISA, Pastorex ® LAT) [12,[18][19][20][21][22] Human serum (paracoccidioidomycosis patients) -Galf -containing glycolipid Paracoccidioides brasiliensis research study [23] Mouse monoclonal Ab (MEST-1)

Aspergillus fumigatus
research study [30] Mouse monoclonal Ab Aspergillus fumigatus research study [31] In addition to Galf -specific antibodies, there is another class of carbohydrate-binding proteins of nonimmune origin, lectins, that can be used as tools for detecting Galf -bearing motifs.Unfortunately, only a few natural lectins have been characterised so far as Galfbinding and have mostly been used in research studies, among which human intelectin-1 (hIntL-1) has been closely studied [32][33][34][35].In addition, all of them do not specifically recognise Galf, but also multiple glycan epitopes engaged in host-pathogen recognition interactions (Table 2).We focus herein on another type of Galf -recognising lectin, neolectin, bioengineered from naturally occuring Galf -ase.First described in 1977 and purified in crude form from the culture supernatants and cell lysates of filamentous fungi, bacteria, and protozoa, it was only in 2015 that an open reading frame (ORF) encoding a Galf -specific enzyme from Gram-positive bacteria Streptomyces sp.(strain JHA19) was identified, cloned, expressed, and completely biochemically characterised [36][37][38][39][40].

Design and Production of Novel Galf-Specific Neolectin (GalfNeoLect)
Our strategy was to generate several mutant variants of the wild-type Galf-ase characterised by attenuated hydrolytic activity towards the pNP β-D-Galf substrate.Since the deduced Galf-ase amino acid sequence analysis, as previously reported [38][39][40]43], revealed that it belongs to the GH2 family, to identify the catalytic residues of interest, we carried out in silico sequence alignment on three bacterial β-galactosidases from the GH2 Figure 1.A graphical abstract depicting the bioengineering of NeoLectins from galactofuranosidase by the site-directed mutagenesis method.The glutamic acid/base catalytic residue (E464) was specifically changed to glutamine (Q), resulting in galactofuranosidase E464Q mutant with attenuated hydrolytic activity that can act as galactofuranose-binding neolectin (Galf NeoLect).

Design and Production of Novel Galf-Specific Neolectin (GalfNeoLect)
Our strategy was to generate several mutant variants of the wild-type Galf -ase characterised by attenuated hydrolytic activity towards the pNP β-D-Galf substrate.Since the deduced Galf -ase amino acid sequence analysis, as previously reported [38][39][40]45], revealed that it belongs to the GH2 family, to identify the catalytic residues of interest, we carried out in silico sequence alignment on three bacterial β-galactosidases from the GH2 family with determined crystal structures.The selected homologous amino acid sequences from Bacillus circulans (PDB accession code 4YPJ), Bacteroides fragilis (PDB accession code 3CMG), and Bacteroides vulgatus (PDB accession code 3GM8), sharing between 24% and 26% of their identities, revealed two sets of absolutely conserved glutamic acid residues (Figure 2, Supplemental Figure S3).A graphical abstract depicting the bioengineering of NeoLectins from galactofuranosidase by the site-directed mutagenesis method.The glutamic acid/base catalytic residue (E464) was specifically changed to glutamine (Q), resulting in galactofuranosidase E464Q mutant with attenuated hydrolytic activity that can act as galactofuranose-binding neolectin (GalfNeoLect).

Design and Production of Novel Galf-Specific Neolectin (GalfNeoLect)
Our strategy was to generate several mutant variants of the wild-type Galf-ase characterised by attenuated hydrolytic activity towards the pNP β-D-Galf substrate.Since the deduced Galf-ase amino acid sequence analysis, as previously reported [38][39][40]43], revealed that it belongs to the GH2 family, to identify the catalytic residues of interest, we carried out in silico sequence alignment on three bacterial β-galactosidases from the GH2 family with determined crystal structures.The selected homologous amino acid sequences from Bacillus circulans (PDB accession code 4YPJ), Bacteroides fragilis (PDB accession code 3CMG), and Bacteroides vulgatus (PDB accession code 3GM8), sharing between 24% and 26% of their identities, revealed two sets of absolutely conserved glutamic acid residues (Figure 2, Supplemental Figure S3).Based on mechanistic grounds of the GH2 family, the conserved residues represent the catalytic nucleophile (E573) and the catalytic acid/base (E464).Identified glutamic acid residues were expected to be the catalytic ones [40] and, therefore, we focused our mutagenesis studies on the acid/base glutamic acid residue (E464) that has a crucial step in glycohydrolitic catalysis.Therefore, the acid/base catalytic residue (E464) has been specifically substituted to alanine (E464A), serine (E464S), cysteine (E464C), and glutamine (E464Q) (Table 3).The acid/base carboxylic acid residue (E464) has been replaced by the amino acids that have the residues of specific interest-briefly, that have no negative charge-and, subsequently, the hydrolytic mechanism rate should be attenuated whilst conserving the substrate specificity, in order to generate and explore their potential role as Galf -specific neolectins (Figure 3A).All of the Galf -ase mutant protein variants, except E464A, were produced and purified under the same conditions as wild-type Galf -ase.It appears that the E464A mutation probably affected the folding and stability of protein, resulting in the formation of insoluble protein aggregates (Figure 3B).Table 3. Codon changes and respective amino acid changes introduced at 464 positions in the wild-type Galf -ase amino acid sequence by site-directed mutagenesis.Color indicates the mutation.

Mutant Variant
Codon Change Amino Acid Change charge-and, subsequently, the hydrolytic mechanism rate should be attenuated whilst conserving the substrate specificity, in order to generate and explore their potential role as Galf-specific neolectins (Figure 3A).All of the Galf-ase mutant protein variants, except E464A ,were produced and purified under the same conditions as wild-type Galf-ase.It appears that the E464A mutation probably affected the folding and stability of protein, resulting in the formation of insoluble protein aggregates (Figure 3B).

Kinetic Characterisation of Galf-Ase Mutant Variants-Identification of Novel Galf-Specific Neolectin (GalfNeoLect)
In analysing the hydrolitic activity and the hydrolysis rate-limiting steps of the three Galf -ase mutant variants E464S, E464C, and E464Q, the Michaelis-Menten kinetic parameters were determined under the similar conditions used for wild-type enzyme [46].All the mutants gave typical Michaelis-Menten saturation curves (Supplemental Figure S4) and exhibited slightly decreased K M values compared to the one determined for wild-type, apart from E464S, which stands out for its sevenfold decrease in the K M value (Table 4).Altogether, the obtained values are in the same order of magnitude.Indeed, the replacement of glutamic acid residue to serine, cysteine, and glutamine has resulted in a 2000-orders-ofmagnitude decrease in the hydrolytic activity compared to the wild-type Galf -ase enzyme, confirming that this residue is essential for catalysis.The significant decrease in catalytic efficiency by a factor of 3000-1400 times for E464S and E464C mutants, respectively, and 56 times lower for E464Q mutant is observed.These kinetic observations are in accordance with the other data obtained in similar assays used for the identification of the catalytic residues in retaining and inverting several GHs [46][47][48].The Galf -ase mutant variant E464Q featured only a minor change in the K M value, 166 µM compared to 250 µM, of the wild-type Galf -ase and, given the advantage that, contrary to other mutant variants, it was purified in sufficient quantities (0.3 mg/L), it was selected as a promising candidate to be tested as a novel Galf -binding lectin (Galf NeoLect).Further in the article, the E464Q mutant variant is addressed as Galf NeoLect.To study and evaluate the binding interactions of Galf -ase E464Q mutant variant, as novel Galf -binding lectin (Galf NeoLect), we synthetised novel neoglycoproteins (NGPs) to serve as the Galf -bearing scaffold.Bovine serum albumin (BSA), a well-known, naturally unglycosylated protein that has up to 60 primary amino functional groups available for click-conjugation chemistry [49], has been functionalised with Galf motifs.Galf -N 3 , involved in the corresponding click reaction with propargyl functionalised BSA, was obtained from the corresponding per-O-acetyl galactofuranose [50] using TMSN 3 as a source of azide and TMSOTf as the Lewis acid (see ESI for chemical synthesis).After acetyl cleavage, the resulting/expected Galf -conjugated BSA neoglycoprotein (Galf NGP) synthesis has been first characterised for its functionality by direct binding assay with human Intelectin-1 (hIntL-1), wild-type Galf -ase, and Galf NeoLect.As hIntL-1 has been reported to bind furanose residues (five-membered-ring saccharide isomers), including a β-Galf -containing disaccharide, it served as a positive control for the evaluation of newly synthetised Galf NGP.The binding ability towards Galf NGP was tested directly by an enzyme-linked lectin assay (ELLA), where the immobilised hIntL-1, wild-type Galf -ase, and Galf NeoLect were incubated in the presence of increasing concentrations of Galf NGP.Under these conditions, the all three tested proteins gave dose-dependent responses (Figure 4) towards Galf NGP with BC 50 (concentration to obtain 50% Galf NGP binding with the selected proteins) values in the same range of magnitude (Table 5).Indeed, BC 50 values at 0.16 µM for Galf NeoLect and hIntL-1 were obtained.These values are comparable and in accordance with the 0.085 ± 0.014 µM functional affinity value obtained with β-D-Galf -substituted surface for hIntL-1 in a previous study [33].These results demonstrate that Galf NGP is a novel and relevant ligand for testing the binding recognition towards Galf.

Profiling GalfNeoLect Specificity
In the competitive inhibition assay, we investigated the ability of selected pNP, azido, or thyoaryl monosaccharide substrates (Galf-N3, Galf-thioaryl, pNP-β-D-Galf, pNP-β-D-Galp, pNP-α-L-Araf, pNP-β-D-Ribf) (Figure 5) for their ability to inhibit the binding of GalfNGP to immobilised GalfNeoLect.The presence of increasing concentrations of Galf -N 3 , Galf -thioaryl, and pNP-β-D-Gal during incubation clearly resulted in a progressive decrease in the fluorescence signals for Galf NeoLect in a dose-dependent manner (Supplemental Figure S5).Indeed, Table 6 summarises the 50% inhibitory concentrations (IC 50 ) obtained from all compounds studied.The lowest IC 50 was obtained for Galf -thioaryl with 0.18 mM, followed by Galf -N 3 with 0.60 mM.On the contrary, feeble responses in terms of detectability and no inhibition were obtained with pNP-β-D-Galp, pNP-α-L-Araf, and pNP-β-D-Ribf monosaccharide substrates (Supplemental Figure S5, and Table 6), which demonstrate their absence of specificity for the tested Galf NeoLectin.These results are in accordance with the IC 50 values (Table 6, Supplemental Figure S6) obtained in the competitive inhibition assay with the wild-type Galf -ase, which interacts with the identical type of inhibitors (Galf -thioaryl) and in the same range of values as shown in a previous study [46].These results demonstrate that the Galf NeoLectin binds specifically to only Galf -motifs and that the site-directed mutagenesis did not affect the substrate specificity.Finally, to test the biological functionality of the Galf NeoLect on crude samples, we used the viable spores from Aspergillus brasiliensis (ATCC 16404) that naturally carry Galfcontaining galactomannan.By inhibition assay, we demonstrate that Aspergillus spores are able to displace the interaction between Galf NGP and Galf NeoLect in a dose-dependent manner (Figure 6).
(Supplemental Figure S5, and Table 6), which demonstrate their absence of specificity for the tested GalfNeoLectin.These results are in accordance with the IC50 values (Table 6, Supplemental Figure S6) obtained in the competitive inhibition assay with the wild-type Galf-ase, which interacts with the identical type of inhibitors (Galf-thioaryl) and in the same range of values as shown in a previous study [44].These results demonstrate that the GalfNeoLectin binds specifically to only Galf-motifs and that the site-directed mutagenesis did not affect the substrate specificity.Finally, to test the biological functionality of the GalfNeoLect on crude samples, we used the viable spores from Aspergillus brasiliensis (ATCC 16404) that naturally carry Galfcontaining galactomannan.By inhibition assay, we demonstrate that Aspergillus spores are able to displace the interaction between GalfNGP and GalfNeoLect in a dose-dependent manner (Figure 6).

Discussion
To bioengineer a galactofuranose-specific neolectin (Galf NeoLect) from a glycoside hydrolase, galactofuranosidase (Galf -ase), we undertook several steps, each followed by a qualitative procedure to determine the following parameters: (a) inactivation rate of hydrolytic activity, (b) conservation of substrate specificity, and (c) probing a robustness Galf -recognition capacity on a novel substrate.In the lack of the available resolved Galf -ase crystal structure, the prediction of the active site amino acid residues was carried out by sequence comparison with the enzymes within the same GH2 family.Based on the sequence identities between 24% and 26% with the Bacillus circulans, Bacteroides fragilis, and Bacteroides vulgatus GHs, two glutamic acid residues, E464 and E573, were highlighted as active site acid/base and nucleophile residues.In the present study, we used the site-directed mutagenesis method for direct confirmation of catalytic function of selected residues.The reduction in the catalytic activities of the mutant enzymes E464S, E464C, and E464Q was consistent with the suggested roles of the altered amino acids as catalytic residues.As indicated by kinetic studies, the catalytic activities of mutant enzymes (k cat /K M ) were 3.000 to 56-fold weaker than that of the wild-type Galf -ase.According to the performance of its kinetic parameters, on-hand quantities, and the fact that, as the amide analogue is substituted to glutamic acid, it does not vary the length of aliphatic chain, the E464Q mutant variant was considered to have satisfying qualities to act as Galf NeoLect.We performed an assessment of whether the change in the catalytic acid/base residue had an impact on the substrate specificity of Galf NeoLect, the variety of pNP, azido, or thyoaryl monosaccharide substrates (Galf -N 3 , Galf -thioaryl, pNP-β-D-Galf, pNP-β-D-Galp, pNP-α-L-Araf, pNP-β-D-Ribf ), including α-L-Araf, a stereochemical analogue of β-D-Galf, differing only by the absence of the extra C-6 hydroxymethyl group.Only three classes of substrates, all having Galf -sugar moiety, showed interaction with Galf NeoLect, bare Galf -N 3, pNP-β-D-Galf and its thioaryl analogue of Galf.Data from competitive inhibition assay reveal that Galf NeoLect recognises multiple Galf -bearing molecules and can discriminate them between other monosaccharides.Also, we synthetised a novel neoglycoprotein based on Galf -decorated bovine serum albumin to investigate whether Galf NeoLect interacts with the more complex structures.BSA has been widely used as scaffold for harbouring multiple carbohydrate motifs via click chemistry [49].The resulting polyvalent Galf NGP mimics carbohydrate presentation at the cell surface and allows one to study the Galf NeoLectcarbohydrate interactions.The Galf -binding capacity of Galf NGP was investigated with hIntL-1, a galactofuranosyl-binding lectin that served as standard, and compared with wild-type Galf -ase and Galf NeoLect in the direct binding assay.The wild-type Galf -ase and Galf NeoLect, together with hIntL-1, showed binding to Galf NGP, indicating that the Galf -moieties are able to trigger the specific interactions with Galf NGP.

Site-Directed Mutagenesis, Overexpression, and Mutant Galf-Ase Protein Purification
A pET-28a(+) plasmid construct encoding the wild-type galactofuranosidase (Galf -ase) gene from JHA 19 Streptomyces sp.[46] was used as a template to generate the mutant Galf -ase genes (E464Q, E464A, E464C, E464S) that were obtained by the site-directed mutagenesis method.The mutagenic primers (Table 7) were designed with Agilent Technologies QuikChange Primer Design tool and the reaction was performed using Agilent Technologies QuikChange Lightning Site-Directed Mutagenesis Kit (Santa Clara, CA, USA).The presence of desired mutations was confirmed by DNA sequencing performed by Eurofins Genomics.The overexpression and purification of mutant Galf -ase proteins was performed following a procedure reported previously [46].Briefly, E. coli Rosetta™ (DE3) (Novagen ® ) expression strain, transformed with the plasmid construct carrying the desired Galf -ase mutant genes, was grown overnight at 37 • C in 1 L Luria-Bertani medium (LB media) supplemented with chloramphenicol (34 µg/mL) and kanamycin (30 µg/mL).Cells were grown to mid-exponential phase (OD 600 : 0.6), then shortly cooled on ice, and protein expression was induced by the addition of β-D-thiogalactopyranoside (IPTG) (100 µL; 1 M), after which the mixture was left on a shaker incubator (250 rpm) for 16 h at 15 • C. Afterwards, cells were harvested by centrifugation (4255× g, 30 min, 4 • C) and the cell pellets were resuspended in lysis buffer solution (1:10 v/v; 100 mM NaCl, 50 mM Tris/HCl pH 8, 1 mM phenylmethanesulfonyl fluoride (PMSF), 5% glycerol, 0.1% Triton X-100, lysozyme 1 mg/L).The suspension was incubated by stirring for 20 min at 4 • C, lysed by three freeze-thaw cycles, and subsequently sonicated on ice.Lysate was centrifuged (30,000 g, 20 min, 4 • C), supernatant was filtered (0.45 µm pore filter), and the protein was then purified from the clarified lysates using pre-equilibrated (10 mL; 50 mM Tris, 200 mM NaCl, 10 mM Imidazole) Thermo Scientific HisPur™ Ni-NTA Chromatography Cartridge (1 mL).The bound protein was eluted by an imidazole gradient (10-500 mM) and an aliquot of eluted fractions was analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8% separating gel, according to Laemmli's method [51], and protein bands were visualised by staining with Coomassie Brilliant Blue G250.Fractions containing pure protein were collected and concentrated by ultrafiltration (30,000 MWCO, Sartorius Vivaspin ® sample concentrator), and protein quantity was determined using colorimetric Bio-Rad protein assay based on the Bradford dye-binding method, with bovine serum albumin (BSA) as a standard.

Direct Binding Assays
The assays were performed according to GlycoDiag's protocol, already described in the literature [49,[52][53][54][55]. Briefly, the neoglycoprotein preliminary, labelled with biotin (in a range of concentrations), was prepared in PBS supplemented with 1 mM CaCl 2 and 0.5 mM MgCl 2 and deposited in each well (50 µL each) of 96-well plates, previously functionalised with either Intelectin (hIntL-1, 9137-IN-050, Biotechne), wild-type Galf -ase, or Galf NeoLect (LEctPROFILE ® plates from GLYcoDiag, Orléans, France) in triplicate and incubated for 2 h at room temperature.After washing with PBS buffer, the streptavidin-DTAF conjugate was added (50 µL) and incubated for 30 min.The plate was then washed again with PBS.Finally, 100 µL of PBS was added for reading the plate using a fluorescence reader (λex = 485 nm, λem = 530 nm, Fluostar OPTIMA, BMG LABTECH, France).The intensity of the signal was directly correlated with the ability of the compound to be recognised by the lectin.

Inhibition Assays
The interaction profiles of each compound were determined through an indirect method, based on the inhibition by the compound of the interaction between a specific coupled lectin-glycan (a neoglycoprotein labelled with biotin and used as a tracer [48]).Briefly, a mix of biotinylated Galf NGP (fixed concentration) and the corresponding compounds (in a range of concentrations), prepared in PBS supplemented with 1 mM CaCl 2 and 0.5 mM MgCl 2 , are deposed in each well (50 µL each) in triplicate and incubated for 2 h at room temperature.After washing with PBS buffer, the conjugate streptavidin-DTAF was added (50 µL) and incubated for 30 min more.The plate was washed again with PBS.
Finally, 100 µL of PBS was added for the readout of the fluorescent plate, performed with a fluorescence reader (λex = 485 nm, λem = 530 nm, Fluostar OPTIMA, BMG LABTECH, France).The signal intensity was inversely correlated with the capacity of the compound to be recognised by the lectin and expressed as inhibition percentage with comparison with the corresponding biotinylated Galf NGP alone.4.9.Preparation of Aspergillus Brasiliensis (ATCC 16404) Spores Aspergillus brasiliensis (ATCC 16404) culture was inoculated on Sabouraud agar plates and incubated at 37 • C for 4 days until the formation of a black mycelium mat, which contains spores.Fresh spores were harvested with a sterile plastic inoculation loop by rubbing the black mycelium mat and dispersed in physiological serum solution (0.8% NaCl, 10 mL).The spore solution was resuspended well by using a vortex to prevent aggregation and was calibrated using a Malassez counting chamber to 5.10 6 spores/mL.Spores' solutions (5.10 6 spores/mL) were used directly in inhibition assays with Galf NeoLect, following the same protocol describe above (Section 4.8).

Conclusions
A natural lectin that specifically binds only a Galf epitope and does not engage with other glycan epitopes has not yet been discovered.Therefore, to the best of our knowledge, we generated the first Galf -specific neolectin from the corresponding wild-type galactofuranosidase that exclusively recognises and interacts with Galf -glycan motifs.Furthermore, we synthetised the novel neoglycoprotein-carrying Galf monosaccharide units and demonstrated that it is recognised (in this study) by all available Galf -binding, hIntL-1, and Galf -specific proteins, as well as wild-type Galf-ase and Galf NeoLect.Furthermore, it may be feasible to exploit the Galf multivalency of Galf -NGP to identify enzymes involved in Galf metabolism and, together with Galf NeoLect, it is an interesting complementary Galforiented system to study host-pathogen interactions or for the qualitative GLYcoPROFILE ® serodiagnosis of infectious diseases such as aspergillosis, leishmaniasis, borreliosis (Lyme disease), or tuberculosis.

Figure 1 .
Figure 1.A graphical abstract depicting the bioengineering of NeoLectins from galactofuranosidase by the site-directed mutagenesis method.The glutamic acid/base catalytic residue (E464) was specifically changed to glutamine (Q), resulting in galactofuranosidase E464Q mutant with attenuated hydrolytic activity that can act as galactofuranose-binding neolectin (GalfNeoLect).

Figure 1 .
Figure1.A graphical abstract depicting the bioengineering of NeoLectins from galactofuranosidase by the site-directed mutagenesis method.The glutamic acid/base catalytic residue (E464) was specifically changed to glutamine (Q), resulting in galactofuranosidase E464Q mutant with attenuated hydrolytic activity that can act as galactofuranose-binding neolectin (GalfNeoLect).

Figure 2 .
Figure 2. Alignment of partial ORF of Galf-ase (Streptomyces spp.) amino acid sequence and corresponding regions of its homologs.The conserved E464 and E573 amino acid residues are indicated with asterisks.

Figure 2 .
Figure 2. Alignment of partial ORF of Galf -ase (Streptomyces spp.) amino acid sequence and corresponding regions of its homologs.The conserved E464 and E573 amino acid residues are indicated with asterisks.

Table 5 .
Comparison of BC50 of GalfNGP towards different Galf-ase-binding proteins and their respective values.

Table 1 .
Comparative summary of antibodies specific for different Galf -containing glycan motifs.

Table 2 .
Comparative summary of natural lectins interacting with different Galf -containing glycan motifs.

Table 3 .
Codon changes and respective amino acid changes introduced at 464 positions in the wildtype Galf-ase amino acid sequence by site-directed mutagenesis.Color indicates the mutation.

Table 4 .
Comparison of Michaelis-Menten kinetics constants for hydrolysis of β-D-Galf by E464XGalf -ases mutant variants.Color indicates the mutation.

Table 5 .
Comparison of BC50 of GalfNGP towards different Galf-ase-binding proteins and spective values.

Table 5 .
Comparison of BC 50 of Galf NGP towards different Galf -ase-binding proteins and their respective values.

Table 7 .
Primers used for site-directed mutagenesis.
a Underlined sequences indicate the substituted codons.
Funding:Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: Data are contained within the article or Supplementary Materials.